Engineering osmolysis susceptibility in Cupriavidus necator and Escherichia coli for recovery of intracellular products

Background Intracellular biomacromolecules, such as industrial enzymes and biopolymers, represent an important class of bio-derived products obtained from bacterial hosts. A common key step in the downstream separation of these biomolecules is lysis of the bacterial cell wall to effect release of cytoplasmic contents. Cell lysis is typically achieved either through mechanical disruption or reagent-based methods, which introduce issues of energy demand, material needs, high costs, and scaling problems. Osmolysis, a cell lysis method that relies on hypoosmotic downshock upon resuspension of cells in distilled water, has been applied for bioseparation of intracellular products from extreme halophiles and mammalian cells. However, most industrial bacterial strains are non-halotolerant and relatively resistant to hypoosmotic cell lysis. Results To overcome this limitation, we developed two strategies to increase the susceptibility of non-halotolerant hosts to osmolysis using Cupriavidus necator, a strain often used in electromicrobial production, as a prototypical strain. In one strategy, C. necator was evolved to increase its halotolerance from 1.5% to 3.25% (w/v) NaCl through adaptive laboratory evolution, and genes potentially responsible for this phenotypic change were identified by whole genome sequencing. The evolved halotolerant strain experienced an osmolytic efficiency of 47% in distilled water following growth in 3% (w/v) NaCl. In a second strategy, the cells were made susceptible to osmolysis by knocking out the large-conductance mechanosensitive channel (mscL) gene in C. necator. When these strategies were combined by knocking out the mscL gene from the evolved halotolerant strain, greater than 90% osmolytic efficiency was observed upon osmotic downshock. A modified version of this strategy was applied to E. coli BL21 by deleting the mscL and mscS (small-conductance mechanosensitive channel) genes. When grown in medium with 4% NaCl and subsequently resuspended in distilled water, this engineered strain experienced 75% cell lysis, although decreases in cell growth rate due to higher salt concentrations were observed. Conclusions Our strategy is shown to be a simple and effective way to lyse cells for the purification of intracellular biomacromolecules and may be applicable in many bacteria used for bioproduction. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-023-02064-8.


Supplementary Tables
*Whole genome sequences of both the parent strain H16 and the evolved strain ht030b were obtained to identify the mutations that arose throughout the ALE. Both genomes were first mapped to the reference C. necator H16 genome obtained by Little et al, 1 and differences between each of the two genomes we sequenced and the reference genomes were identified. Several of these variations were found in both the parent strain and evolved strain, indicating these mutations were not acquired throughout the ALE. Five variations (meeting quality control criteria described in the Methods) were unique to either the parent or evolved strain. Four variations (relative to the reference genome) were found in ht030b, while one was found in the unevolved H16 strain. This is denoted by the column labeled "SNP Present In". Figure 2B in the main text compares the growth of wild-type C. necator H16 with the adapted halotolerant strain ht030b. In that experiment, 4 replicate cultures each of H16 and ht030b were grown in a 24-well plate and grown overnight at 30 ⁰ C, with starting optical densities (A600nm) of 0.01. H16 demonstrated no visible growth, whereas ht030b exhibited exponential growth with a specific growth rate of 0.16 h -1 . However, we have found that growth of H16 in high salt conditions appears dependent on the starting optical density of the culture and other culturing conditions. A similar experiment was therefore performed in 50-mL volumes in 250 mL baffled shake flasks. Both H16 and ht030b were seeded to starting optical densities of ~0.05.

Fig. S1
Growth curve of H16 (blue circles) and ht030b (red diamonds) in LB containing 3.25% NaCl in 50-mL cultures in shake flasks, seeded at an optical density of A600nm=0.05.
Although wild-type H16 did grow slightly in LB containing 32.5 g/L NaCl (final concentration) in this experiment, the evolved strain still grew significantly faster. Calculated specific growth rates were 0.18 h -1 for ht030b and 0.08 h -1 for H16. In addition to the higher starting cell concentration, it is likely that greater oxygen mass transfer was achieved in flasks compared to that in 24-well plates. The growth of H16 is somewhat dependent on the culturing conditions when growing in LB at elevated salt concentrations. However, in all cases, the evolved strain ht030b grew significantly better than the wild-type strain in high salt concentrations.

Fig. S2
Growth Curve of C. necator H16 (blue circles) and H16 ΔmscL (red diamonds) Growth curves were measured for both wild-type Cupriavidus necator H16 and C. necator ΔmscL in LB medium. Overnight cultures of both strains were inoculated to an initial cell density with A600=0.015 in 50-mL cultures in shake flasks. Cultures were grown at 30 ⁰C shaking at 200 rpm for 9 hours, with absorbance measurements (600 nm) taken every 90 minutes.
The growth curves of the two strains in LB were not significantly different. The measured growth rate of the wild-type strain (0.45 ± 0.01 h -1 ) was just slightly higher than the growth rate of the ΔmscL strain (0.43 ± 0.01 h -1 ). Although there was an observable difference between the growth of the two strains, this result was not statistically significant (p>0.07). Therefore, we conclude that the absence of the mscL gene does not significantly affect the growth rate of C. necator, and that the mscL gene is not required for normal functioning of the cell. The maximum salt concentration tolerated by both wild-type C. necator H16 and evolved strain ht030b was determined for both heterotrophic growth (LB) and organoautotrophic growth (M9 formate). To test salt tolerance for heterotrophic growth, both strains were inoculated in 50-mL tubes containing 10 mL LB with variable salt concentrations to a starting OD of 0.001. As the measured average growth rate of C. necator H16 was 0.45 h -1 , we defined the salt tolerance as the maximum salt concentration for which the average growth rate over a 24-hour period exceeded 0.225 h -1 (half of normal growth rate). This corresponded to an optical density of over 0.22 after a 24-hour period.

Supplementary Note 3: Effect of salt concentration on growth of C. necator H16 and C. necator ht030b in LB and M9 Formate
The NaCl concentrations tolerated by H16 and ht030b were 16.3 and 29.4 g/L, respectively. For convenience, NaCl concentrations of 15 g/L and 30 g/L were used for H16 and ht030b respectively for osmolysis experiments of those two strains.
To test NaCl tolerance under formatotrophic growth, various salt concentrations were added to M9 formate (note: these represent the amount of salt added to M9 medium, which already contains various amounts of certain salts, rather than the final salt concentration; final osmolarities are taken into account in the data shown in Figures 3B and 4B in the main text). Both strains (H16 and ht030b) were then inoculated in 50-mL tubes containing 10 mL of formate media to a starting OD of 0.02. Formatotrophic growth in defined medium was significantly slower than heterotrophic growth in rich medium. Optical densities were measured after 48 hours. The optical density threshold for maximum tolerated salt concentration was 0.077, which is half of the measured OD of ht030b after 48 hours in M9 formate with no added salt.
The NaCl concentrations tolerated by H16 and ht030b in M9 formate were 6 g/L and 15 g/L respectively. Therefore, M9 formate with 6 g/L added was used as the growth medium for the experiments described in Figure 3B. For the experiments described in Figure 4B, M9 formate with 16 g/L was used. As shown in Fig. S3D, the drop in cell growth when the added salt concentration is raised from 15 g/L to 18 g/L is fairly small. M9 formate with 16 g/L NaCl added has an osmolarity of 0.834 OsM, which is roughly equivalent to that of a 2.5% NaCl solution. Because osmolysis experiments were performed with salt solutions in 0.5% (w/v) increments, this was a more convenient starting solution from a practical standpoint. Fig S4. Overview of RFP-based cell lysis assay developed. (A) Schematic overview of RFP assay as described in methods. Well-mixed red fluorescence measurements (585 nm excitation/ 620 nm emission) were performed on the well-mixed sample, representing the total RFP content, and from the supernatant following centrifugation, representing the released RFP content. Cell lysis fraction was taken to be the ratio of released RFP to total RFP. (B) Representative linear range validation that was replicated in each experiment to verify that RFP concentration was proportional to fluorescence intensity. (C) Fluorescence intensity measurements of identical RFP-expressing cell samples in various solutions, demonstrating that the fluorescence intensity is not sensitive to the various environments encountered in the assay.

Supplementary Note 4: RFP-Based Cell Lysis Assay Diagram and Measurement Notes
In each osmolysis experiment relying on the RFP-based cell lysis assay described in the main text, samples were verified to ensure they fell within the linear range. Cells expressing RFP following the wash step in the osmolysis protocol were concentrated or diluted such that they were 30%, 60%, 90%, 120%, or 150% of the original cell density. Volumes equivalent to the volume measured in the experiment (usually 150 μL for experiments using C. necator and 50 μL for experiments using E. coli) were aliquoted into a 96-well plate and red fluorescence was measured (same excitation/emission values as described in main text methods). If the standard curve was linear, and all samples measured fell within the linear range, then the osmolysis measurements were considered valid. A representative standard curve is shown in Figure S4B. If needed, samples were further diluted in water such that they did fall within this linear range.
Our assay relies on the assumption that the fluorescent signal is a function only of the concentration of RFP in the sample (i.e., that neither the solvent nor the presence/absence of cells significantly affects the fluorescence measurement). To verify this was always the case, fluorescence measurements were taken on three types of samples encountered throughout the experiments. All samples were prepared from equal volumes of the same culture, and therefore began with same amount of RFP. One sample was resuspended in an aqueous salt solution, and therefore nearly all of the RFP remained within the cell. One sample was resuspended in B-PER™ (a commercial bacterial lysis reagent) and therefore cell membranes were lysed and nearly all the RFP was in solution. In the final sample, cells were resuspended in B-PER™ but were then centrifuged, such that RFP was present in a supernatant free of cell debris. As seen in Fig. S4C, all three samples have nearly identical fluorescence values, within 3% of each other. Therefore, we are confident in assuming that neither the solvent nor the location of RFP with respect to cell biomass significantly impacts fluorescence measurement, and therefore our assay is valid in comparing RFP concentration in the various fractions. As described in the main text, the growth rate of E. coli BL21 ΔmscL ΔmscS was measured to demonstrate a tradeoff between the microbial growth rate and osmolysis efficiency. Growth curves were determined for this strain in LB supplemented with NaCl (if necessary) to final concentrations of 0.5%, 1%, 2%, 3%, and 4% (w/v). Cultures were grown in 50-mL volumes in 250-mL baffled shake flasks at 37 ⁰C, starting at an optical density of 0.01. Absorbance measurements were taken every half hour for cultures grown in 0.5%, 1%, and 2% salt and every hour for cultures grown in 3% and 4% salt. Specific growth rates were calculated from the slope of the line of a semilog plot for the range in which the log of absorbance was linear with respect to time.

Fig. S6
Effect of addition of freeze-thaw step (yellow) with osmolysis for BL21 and BL21 ΔmscL ΔmscS compared to cells only subjected to osmotic downshock (blue).
The effect the adding a freeze-thaw step to osmolysis was determined for BL21 cells grown in LB with 2% NaCl (w/v). The procedure was the same as for other osmolysis experiments with minor modifications. Cells were grown, RFP was expressed, and cells were washed as they were in other BL21 osmolysis experiments. For trials labelled "No Freeze-Thaw" samples were resuspended in distilled water and incubated for 30 min at 30 ºC as was normally done. For samples treated with a freeze-thaw step, however, cells were resuspended in distilled water, placed in a freezer set at −20 ºC for twenty minutes, and then thawed in a heat block set at 37 ºC for ten minutes. Samples from the well-mixed culture and supernatant were taken and measured as they were in previous experiments.
Adding a freeze-thaw step significantly enhances the cell lysis efficiency in BL21 ΔmscL ΔmscS cells. The highest cell lysis (22%) is observed for BL21 ΔmscL ΔmscS cells that are subjected to freeze-thaw, which is roughly 5-fold higher than lysis of BL21 ΔmscL ΔmscS without a freezethaw step and 15-fold higher than lysis of BL21 with a freeze-thaw. This improvement indicates that even higher cell lysis efficiencies may be obtained by combining osmolysis with other methods of cell lysis.
Experiments described in Fig. 5A of the main text were repeated exactly, except with cells grown in LB containing 3% NaCl. Note the considerable difference between osmolytic efficiencies of cells grown in 3% and 4% NaCl. This also allows direct comparison of osmolysis between C. necator ht030b and BL21 (as well as their ΔmscL variants), as they were both grown in 3% NaCl. The percent cell lysis in distilled water following growth in 3% NaCl LB was >90% for ht030b ΔmscL and 14% for BL21 ΔmscL.